Abundances of vitellogenin and heat shock protein 90 during ovarian and embryonic development of Exopalaemon carinicauda.

To evaluate functions of vitellogenin (Vg) and heat shock protein 90 (Hsp90) during ovarian and embryonic development of Exopalaemon carinicauda, the cDNA of Vg (Ec-Vg) was cloned, and the abundances of Ec-Vg and heat shock protein 90 (Hsp90) (Ec-Hsp90) were determined during ovarian and embryonic development. During ovarian development, the concentration of Vg protein in hemolymph was markedly greater than in the ovary and hepatopancreas, and was greatest at sexual maturity. The relative abundance of Ec-Vg mRNA transcript was greatest in the hepatopancreas and almost undetectable in hemocytes. By combining mRNA transcript relative abundances with morphological results, a model was developed to explain the mRNA transcript relative abundance of Ec-Hsp90 and Ec-Vg during ovarian and embryonic development. The relative abundance of Ec-Vg mRNA transcript was greatest during the recovery period after reproduction had occurred (Stage V) and mature stage (Stage IV) in the ovary or hepatopancreas, respectively. There were marked associations of the patterns of Ec-Hsp90 and Ec-Vg mRNA transcript abundances both in hepatopancreas and ovary. During embryonic development, the relative abundance of the two mRNA transcripts were greatest at the metazoea and protozoea stages, respectively. These results indicate that Ec-Vg is produced primarily in the hepatopancreas, secreted into the hemolymph and transported into growing oocytes. It, therefore, is believed that Ec-Vg has an important function in the overall ovarian development and late embryonic development of E. carinicauda. In contrast, Ec-Hsp90 is a regulatory factor for Vg transcription and is important during early organogenesis in E. carinicauda.

CRISPR/Cas9-mediated mutation reveals Pax6 is essential for development of the compound eye in Decapoda Exopalaemon carinicauda.

The compound eye in crustaceans is a main eye type in the animal kingdom, knowledge about the mechanism to determine the development of compound eye is very limited. Paired box protein 6 (Pax6) is generally regarded as a master regulator for eye development. In the present study, a genome-based analysis of the Pax6 gene in the ridge tail white prawn Exopalaemon carinicauda was performed and two members of Pax6 homologs, named Ec-Eyeless (EcEy) and Ec-Twin of eyeless (EcToy) were identified. To understand the function of these two homologs of Pax6 gene in the prawn, the CRISPR/Cas9 genome editing technique was applied to generate EcEy and EcToy knock-out (KO) prawns and their phenotypes were analyzed. The surviving EcEy-KO embryos and larvae exhibited severe abnormal eye morphology, suggesting that EcEy is necessary for the compound eye development in prawn, while no mutant phenotype was found in EcToy-KO individuals. These findings highlighted the conservative role of Pax6 gene in the compound eye formation, and the functional differentiation between EcEy and EcToy gene may reveal a novel regulating mechanism of Pax6 on the compound eye development in the decapods. These data will provide important information for understanding the regulation mechanism for crustacean compound eye development.

Molecular identification and expression profiles of four splice variants of Sex-lethal gene in Cherax quadricarinatus.

Cherax quadricarinatus, as one of the world's most valuable freshwater shrimp species, has received extensive attention in recent years. As males grow larger and faster than females, development of the sex control breeding techniques is of great interest, but knowledge on sex determination and differentiation in C. quadricarinatus remains poorly unknown. Sxl (Sex-lethal) is an important gene in the sexual differentiation regulatory hierarchy. It reflects the ratio of sex chromosomes to autosomes into molecule changes and directs sex-specific splicing forms of precursor mRNA. In the present study, the full-length cDNA sequences of four Sxl splice variants were identified from C. quadricarinatus, designated as CqSxl1, CqSxl2, CqSxl3 and CqSxl4, respectively. Sequence analysis determined different splicing sites near the translation termination region of four Sxl transcript isoforms. Two highly conserved classical RRM domains were found according to predicted secondary structures of Sxl proteins. mRNA expression of CqSxl in different tissues, developmental stage of embryos and testes were investigated by real-time quantitative PCR. Among four isoforms, CqSxl3 showed tissue specificity with higher expression levels in testis than in ovary. CqSxl1 and CqSxl4 were found widely expressed in various tissues and CqSxl2 was almost undetectable. In early developmental stages, the expression levels of CqSxl1/3/4 gradually increased along with embyonic development. In addition, CqSxl genes presented the higher transcript levels in the early stage of testis development. Furthermore, CqSxl3 silencing induced a significant decrease of the transcript of Cq-IAG, an androgenic hormone-encoding gene responsible for masculine development. These data indicate that CqSxl3 might be involved in male sex determination in C. quadricarinatus. Our study will contribute to understanding the mechanism of sex determination in C. quadricarinatus, and also can provide theoretical guidance for establishing a sex control technology.

Embryonic Development in the Peppermint Shrimp, Lysmata boggessi (Caridea: Lysmatidae).

There are a limited number of model species for decapod experimental embryology. To improve our understanding of developmental pattern evolution in the Decapoda, here we describe the early embryonic development of the caridean shrimp Lysmata boggessi, from immediately after fertilization to the hatching of the zoea larva, using fluorescence microscopy and whole-mount nuclear staining with 4',6-diamidino-2-phenylindole. Lysmata boggessi follows the standard caridean pattern of early development, with early holoblastic cleavage that will later become superficial, to form a blastoderm. We found no evidence of stereotypical cleavage and the formation of blastomere interlocking bands, which suggests there is diversity in developmental patterns within the Caridea. Gastrulation starts 37 hours after fertilization, and the embryonized nauplius is formed 2 days later. Enlarged headlobes, early retinal differentiation, and delayed pereopod development are characteristics of the post-naupliar stages in this species. To facilitate comparative studies with other crustacean species, we propose a staging method based on our findings. Lysmata boggessi is a protandric simultaneous hermaphrodite that is relatively easy to breed in captivity and amenable to laboratory experimentation in studies of embryonic development.

Female Growth and Offspring Quality over Successive Spawnings in a Caridean Shrimp Neocaridina davidi (Decapoda, Atyidae) with Direct Development.

This study analyzed the quality of recently spawned eggs and of juveniles over five and six consecutive spawns, respectively, in a caridean shrimp Neocaridina davidi with direct development. The potential energetic antagonism between reproduction and somatic growth was also evaluated. The number of eggs per spawn per female was highest in the first spawn, while the number of recently hatched juveniles per spawn per female declined in the sixth spawn. Lower lipid concentration and energy content were detected in eggs of the fourth and fifth spawns, which may indicate for the first time a decrease in maternal provisioning as a result of multiple spawning in a decapod with direct development. This result had no effect on the size of eggs or of recently hatched juveniles, nor on the growth performance of juveniles during a 30-day period following hatching. Lipids were the most abundant biochemical component of eggs, followed by proteins and glycogen; the relative proportion of each component was probably related to embryonic development type. Egg volume was unsuitable as an indicator of nutrient content, as no correlation was found between these variables. The physiological costs of reproduction were evident from the lower energy content of females that reproduced versus females that remained virgin. The lower body weight of the reproductive females at the end of the experiment showed that allocation of resources to reproduction occurred at the expense of somatic growth. To our knowledge, this is the first empirical demonstration of a decapod with direct development.

In hot and cold water: differential life-history traits are key to success in contrasting thermal deep-sea environments.

Few species of reptant decapod crustaceans thrive in the cold-stenothermal waters of the Southern Ocean. However, abundant populations of a new species of anomuran crab, Kiwa tyleri, occur at hydrothermal vent fields on the East Scotia Ridge. As a result of local thermal conditions at the vents, these crabs are not restricted by the physiological limits that otherwise exclude reptant decapods south of the polar front. We reveal the adult life history of this species by piecing together variation in microdistribution, body size frequency, sex ratio, and ovarian and embryonic development, which indicates a pattern in the distribution of female Kiwaidae in relation to their reproductive development. High-density 'Kiwa' assemblages observed in close proximity to sources of vent fluids are constrained by the thermal limit of elevated temperatures and the availability of resources for chemosynthetic nutrition. Although adult Kiwaidae depend on epibiotic chemosynthetic bacteria for nutrition, females move offsite after extrusion of their eggs to protect brooding embryos from the chemically harsh, thermally fluctuating vent environment. Consequently, brooding females in the periphery of the vent field are in turn restricted by low-temperature physiological boundaries of the deep-water Southern Ocean environment. Females have a high reproductive investment in few, large, yolky eggs, facilitating full lecithotrophy, with the release of larvae prolonged, and asynchronous. After embryos are released, larvae are reliant on locating isolated active areas of hydrothermal flow in order to settle and survive as chemosynthetic adults. Where the cold water restricts the ability of all adult stages to migrate over long distances, these low temperatures may facilitate the larvae in the location of vent sites by extending the larval development period through hypometabolism. These differential life-history adaptations to contrasting thermal environments lead to a disjunct life history among males and females of K. tyleri, which is key to their success in the Southern Ocean vent environment. We highlight the complexity in understanding the importance of life-history biology, in combination with environmental, ecological and physiological factors contributing to the overall global distribution of vent-endemic species.

Spatial variation in the population structure and reproductive biology of Rimicaris hybisae (Caridea: Alvinocarididae) at hydrothermal vents on the Mid-Cayman Spreading Centre.

The dynamics and microdistribution of faunal assemblages at hydrothermal vents often reflect the fine-scale spatial and temporal heterogeneity of the vent environment. This study examined the reproductive development and population structure of the caridean shrimp Rimicaris hybisae at the Beebe and Von Damm Vent Fields (Mid-Cayman Spreading Centre, Caribbean) using spatially discrete samples collected in January 2012. Rimicaris hybisae is gonochoric and exhibits iteroparous reproduction. Oocyte size-frequency distributions (21-823 µm feret diameters) varied significantly among samples. Embryo development was asynchronous among females, which may result in asynchronous larval release for the populations. Specimens of R. hybisae from the Von Damm Vent Field (2294 m) were significantly larger than specimens from the Beebe Vent Field. Brooding females at Von Damm exhibited greater size-specific fecundity, possibly as a consequence of a non-linear relationship between fecundity and body size that was consistent across both vent fields. Samples collected from several locations at the Beebe Vent Field (4944-4972 m) revealed spatial variability in the sex ratios, population structure, size, and development of oocytes and embryos of this mobile species. Samples from the Von Damm Vent Field and sample J2-613-24 from Beebe Woods exhibited the highest frequencies of ovigerous females and significantly female-biased sex ratios. Environmental variables within shrimp aggregations may influence the distribution of ovigerous females, resulting in a spatially heterogeneous pattern of reproductive development in R. hybisae, as found in other vent taxa.

Effect of thermal injury on embryos of banded coral shrimp (Stenopus hispidus) under hypothermal conditions.

Cryopreservation technology regarding banded coral shrimp (Stenopus hispidus) embryos is important as it could improve cultivation and preservation of the species. The development of this technology is to reduce collections of this species from the wild, thus preventing damage to coral reefs. This study investigated the tolerance of different developmental stages of S. hispidus embryos in response to low temperature in the presence or absence of cryoprotectant. Embryos undergoing three stages of embryonic development (eye-formation, heart beat and pre-hatch stage) were selected and exposed to 5, 0 and -5°C in cryoprotectant solutions of 1 or 2M methanol for 1, 2, 4, 6, 8, 16 and 32h. Embryo survival was evaluated based on their hatching percentage. In experiments on the effect of different concentrations of methanol on chilling sensitivity of embryos, it was determined that methanol at 1M methanol reduced the chilling sensitivities of embryos most effectively when compared to the other tested concentrations. Experiments regarding the chilling sensitivity of embryos in different developmental stages indicated that pre-hatch stage embryos were more resistant to subzero temperatures than early stage embryos; they tolerated the 32h exposure at 5 and 0°C without a loss in survival. The study also indicated that late stage embryos are considered to be resistant to chilling, and that pre-late stage embryos are better candidate for cryopreservation.

A histological study of ovarian development in the giant red shrimp Aristaeomorpha foliacea (Crustacea: Decapoda: Aristeidae) from the Southern Tyrrhenian Sea (Western Mediterranean).

The reproductive features of the giant red shrimp, Aristaeomorpha foliacea, were investigated in the southern Tyrrhenian sea by experimental trawl sampling. The annual length-frequency distribution showed a multimodal trend in females, ranging between 16 and 67 mm carapace length (CL), and a unimodal trend in males (18-45 mm CL). Mature males occurred in different proportions all year round, while females displayed seasonal maturity (June-September), with a peak in July. Six oocyte developmental stages were identified, the most advanced of which (Pv, postvitellogenic) had never been described before in this species. Ovary development followed a group-synchronous pattern, with the yolked oocyte stock clearly separated from the reservoir of unyolked oocytes, suggesting that A. foliacea is a total spawner, with determinate fecundity. Based upon histological findings, a revision of macroscopic maturity staging employed in Mediterranean bottom trawl surveys (MEDITS) is proposed.

Lecithotrophic behaviour in zoea and megalopa larvae of the ghost shrimp Lepidophthalmus siriboia Felder and Rodrigues, 1993 (Decapoda: Callianassidae)

Food supply is considered critical for a successful culturing of decapod larvae. However, some species may present yolk reserve sufficient to complete their larval development without external food supply (known as lecithotrophic larval development). In the present study, two experiments were carried out in order to verify whether the callianassid Lepidophthalmus siriboia have lecithotrophic behaviour or, if they need external food for their larval development: Experiment 1, larvae submitted to an initial feeding period and Experiment 2, larvae submitted to an initial starvation period. High survival rate was observed in both experiments, in which only 2 megalopae and 1 zoea III died. These results strongly suggest that larvae of L. siriboia are lecithotrophic as they have sufficiently large yolk reserve to complete their larval development, while the megalopa stage shows facultative lecithotrophy. The larval periods of each stage of the treatments were quite similar and, despite some significant differences in some larval periods, these can be related probably to larval rearing conditions, abiotic factors or, individual variability of larval health, as well as stress caused to the ovigerous females during embryogenesis.

A alimentação é considerada crítica em cultivo de larvas de decápodes. No entanto, algumas espécies podem apresentar reservas nutritivas suficientes para completar o desenvolvimento larval sem a necessidade de alimentação externa (conhecido como desenvolvimento larval lecitotrófico). No presente estudo, dois experimentos foram realizados para verificar se o callianassídeo Lepidophthalmus siriboia tem comportamento lecitotrófico ou se precisa de alimento externo para completar o desenvolvimento larval: Experimento 1, larvas submetidas a um período inicial de alimentação e, Experimento 2, larvas submetidas a um período inicial de inanição. Em ambos os experimentos, observou-se altas taxas de sobrevivência, com apenas 2 megalopas e 1 zoea III mortos. Estes resultados sugerem fortemente que larvas de L. siriboia são lecitotróficas, tendo reservas suficientes para completar o desenvolvimento larval, enquanto o estágio de megalopa apresenta lecitotrofia facultativa. Os períodos larvais de cada estágio nos tratamentos foram bastante semelhantes. No entanto, foram observadas algumas diferenças significantes entre alguns períodos, os quais podem estar relacionados às condições de cultivo, provavelmente devido a fatores abióticos, à variabilidade individual da condição larval, ou ainda a fatores como estresse causado às fêmeas ovígeras durante embriogênese.

Lecithotrophic behaviour in zoea and megalopa larvae of the ghost shrimp Lepidophthalmus siriboia Felder and Rodrigues, 1993 (Decapoda: Callianassidae).

Food supply is considered critical for a successful culturing of decapod larvae. However, some species may present yolk reserve sufficient to complete their larval development without external food supply (known as lecithotrophic larval development). In the present study, two experiments were carried out in order to verify whether the callianassid Lepidophthalmus siriboia have lecithotrophic behaviour or, if they need external food for their larval development: Experiment 1, larvae submitted to an initial feeding period and Experiment 2, larvae submitted to an initial starvation period. High survival rate was observed in both experiments, in which only 2 megalopae and 1 zoea III died. These results strongly suggest that larvae of L. siriboia are lecithotrophic as they have sufficiently large yolk reserve to complete their larval development, while the megalopa stage shows facultative lecithotrophy. The larval periods of each stage of the treatments were quite similar and, despite some significant differences in some larval periods, these can be related probably to larval rearing conditions, abiotic factors or, individual variability of larval health, as well as stress caused to the ovigerous females during embryogenesis.

The importance of feeding in the larval development of the ghost shrimp Callichirus major (Decapoda: Callianassidae).

The present study investigated whether the callianassid Callichirus major shows a lecithotrophic behaviour during larval development. Two experiments were carried out. In the first experiment, larvae were subjected to an initial period of feeding, while in the second they were subjected to an initial period of starvation. In Experiment 1, 80% of C. major larvae succeeded moulting to juvenile stage in treatment with larvae fed every day. In the treatments with larvae fed for 1, 2 and 3 days there was total mortality before they reached the megalopal stage. In Experiment 2, zoea larvae showed more resistance when subjected to an initial period of starvation in which larvae starved for 1, 2 and 3 days and had survival rates of 100, 60 and 80%, respectively. But, a delay in the development duration of the zoeal stages was observed. Total mortality was observed for larvae reared in the treatment with entire starvation. The results suggest that zoeal stages of C. major are not lecithotrophic.

The importance of feeding in the larval development of the ghost shrimp Callichirus major (Decapoda: Callianassidae)

The present study investigated whether the callianassid Callichirus major shows a lecithotrophic behaviour during larval development. Two experiments were carried out. In the first experiment, larvae were subjected to an initial period of feeding, while in the second they were subjected to an initial period of starvation. In Experiment 1, 80 percent of C. major larvae succeeded moulting to juvenile stage in treatment with larvae fed every day. In the treatments with larvae fed for 1, 2 and 3 days there was total mortality before they reached the megalopal stage. In Experiment 2, zoea larvae showed more resistance when subjected to an initial period of starvation in which larvae starved for 1, 2 and 3 days and had survival rates of 100, 60 and 80 percent, respectively. But, a delay in the development duration of the zoeal stages was observed. Total mortality was observed for larvae reared in the treatment with entire starvation. The results suggest that zoeal stages of C. major are not lecithotrophic.

O presente estudo investigou se o callianassid Callichirus major apresenta um comportamento lecitotrófico durante o desenvolvimento larval. Dois experimentos foram realizados. No primeiro experimento, larvas foram submetidas a um período inicial de alimentação, enquanto no segundo elas foram submetidas a um período inicial de inanição. No experimento 1, 80 por cento das larvas de C. major mudaram com sucesso para o estágio de juvenil no tratamento com larvas alimentadas diariamente. Nos tratamentos com larvas alimentadas por 1, 2 e 3 dias, houve uma mortalidade total antes de alcançarem o estágio de megalopa. No experimento 2, as larvas zoés mostraram mais resistência quando submetidas a um período inicial de inanição. Nos tratamentos nos quais as larvas estiveram em inanição por 1, 2 e 3 dias, as taxas de sobrevivência foram100 por cento, 60 por cento e 90 por cento, respectivamente. Porém atrasos na duração do desenvolvimento dos estágios de zoés foram observados. Houve mortalidade total para as larvas cultivadas no tratamento com ausência constante de alimento. Os resultados sugerem não existir um comportamento lecitotrófico nas zoés de C. major.

Mesendoderm cells induce oriented cell division and invagination in the marine shrimp Sicyonia ingentis.

The mesendoderm (ME) cells are the two most vegetal blastomeres in the early developing embryo of the marine shrimp Sicyonia ingentis. These two cells enter mitotic arrest for three cycles after the 5th cell cycle (32-cell stage) and ingress into the blastocoel at the 6th cycle (62-cell stage). Circumjacent to the ingressing ME cells are nine presumptive naupliar mesoderm (PNM) cells that exhibit a predictable pattern of spindle orientation into the blastopore, followed by invagination. We examined the role of ME cells and PNM cells in gastrulation using blastomere recombinations and confocal microscopy. Removal of ME progenitors prevented gastrulation. Removal of any other blastomeres, including PNM progenitors, did not interfere with normal invagination. Altered spindle orientations occurred in blastomeres that had direct contact with one of the ME cells; one spindle pole localized to the cytoplasmic region closest to ME cell contact. In recombined embryos, this resulted in an extension of the region of ME-embryo contact. Our results show that ME cells direct the spindle orientations of their adjacent cells and are consistent with a mechanism of oriented cell division being a responsible force for archenteron elongation.

Early embryonic development of the central nervous system in the Australian crayfish and the Marbled crayfish (Marmorkrebs).

This study sets out to provide a systematic analysis of the development of the primordial central nervous system (CNS) in embryos of two decapod crustaceans, the Australian crayfish Cherax destructor (Malacostraca, Decapoda, Astacida) and the parthenogenetic Marbled crayfish (Marmorkrebs, Malacostraca, Decapoda, Astacida) by histochemical labelling with phalloidin, a general marker for actin. One goal of our study was to examine the neurogenesis in these two organisms with a higher temporal resolution than previous studies did. The second goal was to explore if there are any developmental differences between the parthenogenetic Marmorkrebs and the sexually reproducing Australian crayfish. We found that in the embryos of both species the sequence of neurogenetic events and the architecture of the embryonic CNS are identical. The naupliar neuromeres proto-, deuto-, tritocerebrum, and the mandibular neuromeres emerge simultaneously. After this "naupliar brain" has formed, there is a certain time lag before the maxilla one primordium develops and before the more caudal neuromeres follow sequentially in the characteristic anterior-posterior gradient. Because the malacostracan egg-nauplius represents a re-capitulation of a conserved ancestral information, which is expressed during development, we speculate that the naupliar brain also conserves an ancestral piece of information on how the brain architecture of an early crustacean or even arthropod ancestor may have looked like. Furthermore, we compare the architecture of the embryonic crayfish CNS to that of the brain and thoracic neuromeres in insects and discuss the similarities and differences that we found against an evolutionary background.

Stages and other aspects of the embryology of the parthenogenetic Marmorkrebs (Decapoda, Reptantia, Astacida).

The early development of the parthenogenetic Marmorkrebs (marbled crayfish) is described with respect to external morphology, cell lineage, and segment formation. Due to its parthenogenetic reproduction mode, the question arises whether or not the marbled crayfish is a suitable model organism for developmental approaches. To address this question, we describe several aspects of the embryonic development until hatching. We establish ten stages based on characteristic external changes in the living eggs such as blastoderm formation, gastrulation process, formation and differentiation of the naupliar and post-naupliar segments, limb bud differentiation, and eye differentiation. The study of the post-naupliar cell division patterns, segment formation, and engrailed expression reveals distinct similarities to that of other freshwater crayfish. On this basis, we evaluate the possibility of a generalization of ontogenetic processes in the Marmorkrebs for either freshwater crayfish or other crustacean developmental systems.

Ontogeny of the antennal glands in the crayfish Astacus leptodactylus (Crustacea, Decapoda): immunolocalization of Na+,K+-ATPase.

The involvement of the antennal urinary glands in the ontogeny of osmoregulatory functions was investigated during the development of Astacus leptodactylus by measurements of hemolymph and urine osmolality in juvenile and adult crayfish and by the immunodetection of the enzyme Na+,K+-ATPase. In stage II juveniles, 1-year-old juveniles, and adults, all of which were maintained in freshwater, urine was significantly hypotonic to hemolymph. In adults, chloride and sodium concentrations were much lower in urine than in hemolymph. During embryonic development, Na+,K+-ATPase was detected by immunocytochemistry in ionocytes lining the tubule and the bladder, at an eye index (EI) of 220-250 microm, and in the labyrinth, at EI 350 microm. In all regions, immunofluorescence was mainly located at the basolateral side of the cells. No immunofluorescence was detected at any stage in the coelomosac. In late embryonic stages (EI 410-440 microm), in stage I juveniles, and in adults, strong positive immunofluorescence was found from the labyrinth up to and including the bladder. These results show that, as early as hatching, juvenile crayfish are able to produce dilute urine hypotonic to hemolymph. This ability originates from the presence of Na+,K+-ATPase in ion-transporting cells located in the labyrinth, the tubule, and the bladder of the antennal glands and constitutes one of the main adaptations of crayfish to freshwater.

Effect of temperature on lipids, proteins, and carbohydrates levels during development from egg extrusion to juvenile stage of Cherax quadricarinatus (Decapoda: Parastacidae).

The influence of temperature on biochemical composition, survival and duration of development of Cherax quadricarinatus from egg extrusion to juvenile was analyzed. Berried females were individually subjected to each of 22, 25, 28 and 31 degrees C (n=5 per temperature). Egg samples were obtained every 3 days from egg extrusion to juvenile stage for biochemical analysis. Duration of development and survival decreased with increasing temperature. At 22 and 25 degrees C half of the initial lipid content was consumed during development. At 28 and 31 degrees C, 80% of the initial amount of lipids was consumed. For proteins, depletion rate was significantly lower at 25 degrees C (36% of the initial amount) than at 22, 28 and 31 degrees C (61-65% of the initial amount). For carbohydrates, a significant consumption was observed only at 22 degrees C. Total energy consumption was lower at 22 and 25 degrees C than at 28 and 31 degrees C. We conclude that 22-25 degrees C is the optimal temperature range for C. quadricarinatus egg incubation, although 25 degrees C might be better in terms of development duration in terms of survival, energy cost and protein consumption.

Use of the confocal laser scanning microscope in studies on the developmental biology of marine crustaceans.

Confocal Laser Scanning Microscope techniques have been applied to study the developmental biology of marine copepods and decapod larvae. The lipophylic probes DiI and DiOC(6) were used to study both the external and internal morphology of these crustaceans, whereas the same DiOC(6) and the specific nuclear probe Hoechst 33342 were used to study embryonic development of copepods in vivo. To distinguish viable from non-viable copepod embryos, the vital dye dichlorodihydrofluorescein diacetate (H(2)DCFDA) was used. Major advantages and difficulties in the use of these non-invasive techniques in studies of the reproductive biology of marine crustaceans are discussed.

Isolation of a cDNA encoding a putative SPARC from the brine shrimp, Artemia franciscana.

SPARC (Secreted protein, acidic, rich in cysteine) is an extracellular matrix-associated and anti-adhesive glycoprotein extensively studied in vertebrates. Its presence among invertebrates has been reported in nematodes and flies. We cloned a cDNA containing a complete open reading frame for SPARC from the brine shrimp, Artemia franciscana. The amino acid sequence identity between the Artemia and the fly SPARCs was 55%, whereas that of the Artemia and the nematode proteins was 45%. Artemia and vertebrates exhibited a sequence identity of 30% in the predicted aa sequences. The SPARC consisted of four domains commonly found among reported SPARCs. The protein comprised 291 amino acids, having a signal peptide, a follistatin-like domain, one N-glycosylation site and one calcium-binding EF-hand motif. Fourteen cysteine residues conserved among all the secreted forms of SPARCs were present in the Artemia SPARC, and four extra cysteine residues were also found in it. The extra residues were conserved among SPARCs of the arthropods and the nematode. Phylogenetic analyses showed that the sequences of SPARCs were grouped into those of vertebrates and invertebrates. Though the structural organization of SPARC was conserved among all the species studied, SPARC within a group was highly conserved within that group, but divergent between the two. Northern blots revealed the presence of a 1.1 kb mRNA, which was faintly expressed in embryos and considerably detected in prenauplii and nauplii. The isolation of a SPARC cDNA from Artemia franciscana provides intriguing features of the divergent protein, SPARC.